A key step in the generation of cell lines producing recombinant proteins is the selection of viable clones following the incorporation of the gene of interest into the host cell. For industrial-scale bio-production, a clone in which the product gene is stably integrated and yields a high level of the protein product is highly desired.
In a heterogeneous population, the integration of a recombinant gene into the host genome is a largely random event. The proportion of cells containing multiple copies of stably integrated genes would be small compared to those with low copy numbers. The high-producing subclones are rare and tend to be diluted out by the faster growing non- or low-producing cells. Thus, to isolate a subclone with an increased production rate, many wells would need to be screened and tested. In general, limited dilution methods are tedious and time consuming.
The advent of selection methods that use flow cytometry and cell sorting considerably increased the number of cells that can be screened. Several million cells can be screened in a short time, and subpopulations and single cells can be isolated from within mixed-cell populations even when they are present at frequencies as low as 10−6 within the population.
Flow cytometry was partnered with a non-fluorescent reporter protein for rapid, early stage identification of clones producing high levels of a target protein. This has been facilitated by the availability of antibody and ligand-conjugated fluorochromes enabling isolation of cells based on cell-surface protein expression. For example, a cell surface protein, not normally expressed on host cells, may be co-expressed, with the target protein as a reporter.
In the absence of a correlation between surface expression and productivity, cells can be isolated based on levels of intracellular proteins using reporter molecules such as green fluorescent protein (GFP). The GFP has become an important reporter for gene expression and the selection of cells based on inducible gene products. In mammalian cell lines, GFP has been used for the selection of high-producing clones by co-expression with recombinant proteins and selection based on fluorescence intensity. A correlation between GFP fluorescence intensity and recombinant protein production has been seen for several cell lines expressing various recombinant proteins. However, expression of these selection markers increases the load on the protein expression machinery in the cells and reduces the production of the target protein.